Effects of ICI 182, 780, an ERα and ERß antagonist, and G-1, a GPER agonist, on autophagy in breast cancer cells / Efeitos do antagonista seletivo do REα e do REß ICI 182, 780 e do agonista de GPER G-1 no processo de autofagia em células de câncer de mama

ABSTRACT Objective To investigate if ICI 182,780 (fulvestrant), a selective estrogen receptor alpha/beta (ERα/ERβ) antagonist, and G-1, a selective G-protein-coupled receptor (GPER) agonist, can potentially induce autophagy in breast cancer cell lines MCF-7 and SKBr3, and how G-1 affects cell viability. Methods Cell viability in MCF-7 and SKBr3 cells was assessed by the MTT assay. To investigate the autophagy flux, MCF-7 cells were transfected with GFP-LC3, a marker of autophagosomes, and analyzed by real-time fluorescence microscopy. MCF-7 and SKBr3 cells were incubated with acridine orange for staining of acidic vesicular organelles and analyzed by flow cytometry as an indicator of autophagy. Results Regarding cell viability in MCF-7 cells, ICI 182,780 and rapamycin, after 48 hours, led to decreased cell proliferation whereas G-1 did not change viability over the same period. The data showed that neither ICI 182,780 nor G-1 led to increased GFP-LC3 puncta in MCF-7 cells over the 4-hour observation period. The cytometry assay showed that ICI 182,780 led to a higher number of acidic vesicular organelles in MCF-7 cells. G-1, in turn, did not have this effect in any of the cell lines. In contrast, ICI 182,780 and G-1 did not decrease cell viability of SKBr3 cells or induce formation of acidic vesicular organelles, which corresponds to the final step of the autophagy process in this cell line. Conclusion The effect of ICI 182,780 on increasing acidic vesicular organelles in estrogen receptor-positive breast cancer cells appears to be associated with its inhibitory effect on estrogen receptors, and GPER does notseem to be involved. Understanding these mechanisms may guide further investigations of these receptors' involvement in cellular processes of breast cancer resistance.

RESUMO Objetivo Avaliar o efeito dos compostos ICI 182,780 (fulvestranto), um antagonista seletivo dos receptores de estrógeno alfa/beta (REα/REβ), e do G-1, um agonista seletivo de receptores de estrógeno acoplados a proteínas-G (GPER), na possível indução de autofagia em linhagens de câncer de mama MCF-7 e SKBr3, bem como o efeito de G-1 na viabilidade celular. Métodos A viabilidade celular de células MCF-7 e SKBr3 foi avaliada pelo ensaio com MTT. Para investigar a indução da autofagia, células MCF-7 foram transfectadas com GFP-LC3, um marcador de autofagossomos, e analisadas por microscopia de fluorescência em tempo real. As células MCF-7 e SKBr3 foram incubadas com o indicador de compartimentos ácidos laranja de acridina e analisadas por citometria de fluxo como indicativo para autofagia. Resultados Em células MCF-7, o ICI 182,780 e rapamicina após 48 horas levaram à diminuição da viabilidade celular, enquanto o G-1 não alterou a viabilidade no mesmo período de tratamento. Nem o ICI 182,780 e nem o G-1 induziram aumento na pontuação de GFP-LC3 em células MCF-7 até 4 horas. Já os ensaios de citometria de fluxo demonstraram que ICI 182,780 levou ao aumento de compartimentos ácidos em células MCF-7. O G-1 não aumentou estes parâmetros em ambas as linhagens. Por outro lado, ICI 182,780 e G-1 não induziram à redução da viabilidade em células SKBr3 e nem à formação de compartimentos ácidos, como etapa final do processo autofágico. Conclusão O aumento de compartimentos ácidos pelo ICI 182,780 em células de câncer de mama positivas para receptores de estrógeno parece estar associado com seu efeito inibidor de receptores de estrógeno, mas sem o envolvimento de GPER. A compreensão desses mecanismos pode direcionar estudos sobre o envolvimento dos receptores nos processos celulares de resistência do câncer de mama.

Anthropometric indicators of obesity in the prediction of high body fat in adolescents / Indicadores antropométricos de obesidade na predição de gordura corporal elevada em adolescentes

OBJECTIVE: To determine the anthropometric indicators of obesity in the prediction of high body fat in adolescents from a Brazilian State. METHODS: The study included 1,197 adolescents (15-17 years old). The following anthropometric measurements were collected: body mass (weight and height), waist circumference and skinfolds (triceps and medial calf). The anthropometric indicators analyzed were: body mass index (BMI), waist circumference (WC), waist-to-height ratio (WHtR) and conicity index (C-Index). Body fat percentage, estimated by the Slaughter et al equation, was used as the reference method. Descriptive statistics, U Mann-Whitney test, and ROC curve were used for data analysis. RESULTS: Of the four anthropometric indicators studied, BMI, WHtR and WC had the largest areas under the ROC curve in relation to relative high body fat in both genders. The cutoffs for boys and girls, respectively, associated with high body fat were BMI 22.7 and 20.1kg/m², WHtR 0.43 and 0.41, WC 75.7 and 67.7cm and C-Index 1.12 and 1.06. CONCLUSIONS: Anthropometric indicators can be used in screening for identification of body fat in adolescents, because they are simple, have low cost and are non-invasive. .

OBJETIVO: Determinar os indicadores antropométricos de obesidade na predição da gordura corporal elevada em adolescentes de um estado brasileiro. MÉTODOS: O estudo incluiu 1.197 adolescentes (15-17 anos). As seguintes medidas antropométricas foram coletadas: massa corporal e estatura, perímetro da cintura e dobras cutâneas (tríceps e perna medial). Os indicadores antropométricos analisados foram: índice de massa corporal (IMC), perímetro da cintura (PC), razão cintura-estatura (RCE) e índice de conicidade (IC). A gordura corporal elevada, estimada pela equação de Slaughter et al., foi usada como método de referência. Estatística descritiva, teste U de Mann-Whitney e curva ROC foram usadas para a análise dos dados. RESULTADOS: Dos quatro indicadores antropométricos estudados, o IMC, a RCE e o PC tiveram as maiores áreas sob a curva ROC em relação à gordura corporal elevada relativa em ambos os sexos. Os pontos de corte para os rapazes e as moças, respectivamente, associados com gordura corporal elevada foram IMC 22,7 e 20,1 kg/m2, RCE 0,43 e 0,41, PC 75,7 e 67,7 cm e IC 1,12 e 1,06. CONCLUSÕES: Os indicadores antropométricos podem ser usados como ferramenta para identificação da gordura corporal em adolescentes, por serem um método simples, de baixo custo e não invasivo. .

Family A G-Protein coupled receptors: an overview of structure-function relationships

Family A G-protein coupled receptors (AGPCRs) form the largest group of correlate receptors whose structure, a bundle of seven-trans-membrane (7 TM) helices, may be activated thus becoming able to transduce a signal from the extracellular medium to the cytosol. This activation may be constitutional, for instance due to permanent structural modifications, or be physiologically triggered by agonist binding at an external and accessible specific site. Based on thestructures of agonists, AGPCRs may be divided according to pharmacological assays into many classes of receptors, each one comprising many types or sub-types of proteins, as differentiated by specific binding of inhibitors, all of them performing a multitude of functions. It is noteworthy that AGPCRs have been more recently cloned and their sequences of amino acids determined in a large scale, a condition that has allowed these receptors to be sorted by a new criterium. Sequence analyses have consistently matched functional assays for classification of AGPCRs except for a certain number of functionally unknown receptors which have been cataloged as orphan receptors. A colossal number of AGPCRs, more than 10,000 sequences belonging to more than 1,000 different types of receptors, may nowadays be multiply-aligned what has been enabling the determination of parameters of residue conservation and characterization of special motifs along the structure of these proteins. There are at the present time, high-resolution 3D structures for the following AGPCRs: inactive rhodopsin, retinal-free opsin, Beta adrenoceptor and adenosine receptors. Among them, hodopsin structures are reliable enough to be used as prototypes for analyses of residue conservation and mechanisms of activation of receptors, specially at the level of the more conserved structure in the cytosolic half of their 7TM bundle.